The present invention refers to a method for inducing tumor apoptosis by influencing the ROS (reactive oxygen species) signaling pathway in tumor cells. Increasing the level of ROS leads to the selective inactivation of a tumor cell catalase and subsequently to an apoptosis of these cells. The level of ROS can be increased by increasing the level of nitric oxide through inhibition of the enzymes nitric oxide dioxygenase or arginase. According to the present invention inhibitors of the nitric oxide dioxygenase or arginase can be used for the manufacture of a medicament for the treatment of cancer. The present invention further provides a method for identifying compounds which can be used for the treatment of cancer, wherein the method allows to specifically identify compounds which induce apoptosis through the ROS signaling pathway. The present invention also provides a kit for identifying compounds which induce tumor apoptosis by inactivating a catalase on the tumor cell surface.
WO 2005/117545 discloses that NO catabolic pathways may provide immune resistance to carcinomas, and thus serve as novel targets for cancer intervention. According to this document, the NO catabolic pathways can be influenced by using nitric oxide dioxygenase inhibitors in an amount sufficient to increase the intra-cellular concentration of nitric oxide (NO). However, WO 2005/117545 does not disclose the selective tumor cell apoptosis by destroying the tumor cell catalase and mediating the ROS signaling pathway. The present invention is not based on an immunological effect of nitric oxide dioxygenase inhibitors but on the selective destruction of tumor cells.
WO 2005/000208 refers to the use of a combination of an HMG-CoA reductase inhibitor and an azole for the treatment of a neoplasm. The use of a combination of those two compounds was based on the finding that azoles enhance the anti-proliferative activity of HMG-CoA reductase inhibitors against cancer cells in vitro.
Furthermore, WO 2004/073623 refers to a method of treating a subject having elevated arginase as a symptom or cause of a disorder. Such a treatment comprises administering an arginase inhibitor in order to enhance the arginine bioavailability in the subject. However, there is no teaching in this document that arginase inhibitors can be used to destroy tumor cell catalase and induce an tumor cell selective cell apoptosis.
WO 03/03990 refers to the use of a compound that decreases the production of a protein involved in arginine metabolism in the preparation of a medicament for treating asthma or allergies. Any effect of arginase inhibitors on the tumor cell catalase or the ROS signaling pathway is not described in this document.
Buga et al. (Am. J. Physiol., 1989, pages 1256-1264) describe the use of NG-hydroxy-L-arginine (NOHA) for inhibiting Caco-2 tumor cell proliferation. The effect of arginase inhibitors on selective tumor cell apoptosis as used in the present invention is not indicated in this document.
WO 97/05873 also discloses the use of fluconazole (a nitric oxide dioxygenase inhibitor) for the inhibition of the growth of tumor cells due to the properties of fluconazole to potentiate the effect of a chemotherapeutic agent. Similarly, U.S. Pat. No. 5,565,478 refers to the augmentation of the activity of paclitaxel by ketoconazole when used for the treatment of cancer.
Furthermore, DE 199 63 052 discloses the use of azoles for the prevention of skin cancer, mediated by UV-radiation. None of the above documents discloses a selective tumor cell apoptosis. Especially, there is no teaching to use a combination of an nitric oxide dioxygenase inhibitor and an arginase inhibitor for the treatment of cancer.
It is furthermore known from the experimental work of Irani et al. (Science 1997; 275:1649-1652) that oncogenic transformation of cells causes constitutive generation of extracellular superoxide anions through a membrane associated NADPH oxidase. In the following, tumor cells are distinguished from transformed cells. Transformed cells can be defined as cells which have been transformed in vitro, either by expression of specific oncogenes, by chemical or physical carcinogenic changes or spontaneous transformation, and possess potential for tumor formation. Tumor cells as well as transformed cells produce extracellular superoxide anions. The superoxide anions react to hydrogen peroxide in a close distance to the cell surface. Hydrogen peroxide is long-lived and far-ranging, but nevertheless much more reactive than superoxide anions. For example, superoxide anions do not induce apoptosis directly, but hydrogen peroxide does, if a certain concentration is reached.
Furthermore it is known from Engelmann and Bauer (Anticancer Research 2000; 20:2297-2306) that not transformed fibroblasts, which have been stimulated with TGF (transforming growth factor)-beta or FGF (fibroblast growth factor), can serve as a intercellular effector cell and induce apoptosis to transformed cell. Not transformed fibroblasts are not damaged.
It is known from Bauer et al. (Prostaglandines, Leukotrienes and Essential Fatty Acids 2002; 66:41-56) that the transformed cells release superoxide anions, which exhibit signaling functions such as regulation of proliferation and maintenance of the transformed state. The dismutation product hydrogen peroxide regulates the intracellular level of catalase, whose activity has been observed to be upregulated in certain transformed cells. After glutathione depletion, the transformed cell-derived reactive oxygen species (ROS) exhibit apoptosis-inducing potential through the metal-catalyzed Haber-Weiss reaction, which turns hydrogen peroxide into the highly reactive, extremely short-lived and short-ranging hydroxyl radical from.
Moreover, transformed cell-derived ROS represent key elements for selective and efficient apoptosis induction by natural antitumor systems (such as fibroblasts, granulocytes and macrophages). These effector cells release peroxidase, which utilizes target cell-derived hydrogen peroxide for HOCl synthesis. In a second step, HOCl interacts with target cell-derived superoxide anions and forms apoptosis-inducing hydroxyl radicals (HOCl pathway). In a parallel signaling pathway, effector cell-derived NO interacts with target cell-derived superoxide anions and generates the apoptosis inducer peroxynitrite. Therefore, transformed cell-derived ROS determine transformed cells as selective targets for induction of apoptosis by these effector systems. It was therefore proposed that transformed cell-derived ROS interact with associated cells to exhibit directed and specific signaling functions, some of which are beneficial and some of which can become detrimental to transformed cells.
WO 2005/056048 discloses that superoxide anions react with nitric oxide and produce the highly reactive peroxynitrite in close distance to the transformed cells. Furthermore tumor cells comprise a catalase on the cell surface which deactivates peroxynitrite and therefore inhibits apoptosis. It was found that the inhibition of this catalase consequently prevents the decomposition of peroxynitrite and leads to apoptosis in tumor cells.
WO 2005/056048 furthermore discloses that the generation of hydrogen peroxide from superoxide anions on the surface is not sufficient to induce apoptosis in these cells. As described above, the hydrogen peroxide is transformed into hypochlorous acid by a peroxidase and the subsequent interaction of hypochlorous acid with superoxide anions results in the formation of hydroxyl radicals. It was found that the inhibition of a catalase of the tumor cells which also reacts with hydrogen peroxide leads to a higher concentration of hydrogen peroxide and hydroxyl radicals respectively. The increased levels of apoptosis inducers subsequently lead to a selective apoptosis in the tumor cells. WO 2005/056048 therefore provides a method for specifically increasing the sensitivity of tumor cells against apoptosis by inhibiting the catalase of the tumor cells.